High-antifouling Polymer Brush Coatings on Nonpolar Surfaces via

Research Article Cite This: ACS Appl. Mater. Interfaces 2017, 9, 44281−44292

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High-Antifouling Polymer Brush Coatings on Nonpolar Surfaces via Adsorption-Cross-Linking Strategy Leixiao Yu,† Yong Hou,† Chong Cheng,† Christoph Schlaich,† Paul-Ludwig Michael Noeske,§ Qiang Wei,*,†,‡,∥ and Rainer Haag*,†,∥ †

Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany Department of Cellular Biophysics, Max-Planck Institute for Medical Research, Heidelberg, Heisenbergstr. 3, 70569 Stuttgart, Germany § Fraunhofer Institute for Manufacturing Technology and Advanced Materials IFAM, Wiener Str. 12, 28359 Bremen, Germany ∥ Multifunctional Biomaterials for Medicine, Helmholtz Virtual Institute, Kantstr. 55, 14513 Teltow-Seehof, Germany

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‡

S Supporting Information *

ABSTRACT: A new “adsorption-cross-linking” technology is presented to generate a highly dense polymer brush coating on various nonpolar substrates, including the most inert and low-energy surfaces of poly(dimethylsiloxane) and poly(tetrafluoroethylene). This prospective surface modification strategy is based on a tailored bifunctional amphiphilic block copolymer with benzophenone units as the hydrophobic anchor/chemical cross-linker and terminal azide groups for in situ postmodification. The resulting polymer brushes exhibited long-term and ultralow protein adsorption and cell adhesion benefiting from the high density and high hydration ability of polyglycerol blocks. The presented antifouling brushes provided a highly stable and robust bioinert background for biospecific adsorption of desired proteins and bacteria after secondary modification with bioactive ligands, e.g., mannose for selective ConA and Escherichia coli binding. KEYWORDS: polymer brush coating, adsorption-cross-linking, low-energy surface modification, low fouling, biospecific surface

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for material surface modification,8 it is still difficult to construct a dense polymer brush coating on nonpolar polymeric surfaces, e.g., polyolefines, such as polystyrene (PS) and extremely unreactive surfaces, i.e., poly(dimethylsiloxane) (PDMS) and poly(tetrafluoroethylene) (PTFE), due to the lack of functional surface groups. State-of-the-art methods include direct brush formation via plasma irradiation,9 physisorption of amphiphiles,10 and activation of the substrate by denatured proteins,11 or a layer of dopamine/polyphenol coating12,13 followed by brush generation. However, there are still unresolved challenges from the point of view of technical applicability. Plasma treatment may not only tailor the properties of thin coatings but may also change the surface properties of the underlying substrates, and it requires sophisticated equipment. Also, the traditional amphiphilic coatings suffer from stability problems when exposed to liquid medium for several days. The so-called universal polydopamine/polyphenol or catecholic coatings are not stable enough on nonpolar surfaces, and they also dramatically increase the thickness and roughness of the substrates, whereas denatured proteins often undergo degradation in physiological environments. Moreover, the greatest

INTRODUCTION With the fast development and diversification in biomaterial science, there is an increasing utilization of polymeric materials, especially for implant devices, blood-contacting devices, and biosensors.1 Polymeric biomaterials used for these purposes are mostly selected on the basis of their bulk mechanical properties, rather than the suitability of their surface properties. However, the adsorption of blood proteins on the surfaces initiates a cascade of biological responses and also hinders the effectiveness of the internal body-attached sensoric or implant devices.2−4 Therefore, surface modification of polymeric biomaterials has been of intense recent interest. Some types of surface modification only require a relatively low density of immobilized functional groups; for example, triggering cell spreading just needs a minimum density of as low as 1 fmol/ cm2 adhesive ligands.5 Unlike these, a protein-resistant surface strictly requires a high coverage ratio of the coatings, allowing very limited defect densities or rather no defects.6 Ideally, the coatings should be thin, smooth, and colorless to maintain the bulk or sensoric properties of the materials to the least possible extent. Polymer brushes, which are ultrathin and consist of polymer chains that are tethered with one chain end to an interface,7 are extremely suitable to become protein-resistant coatings. Although there is a vast body of literature regarding strategies © 2017 American Chemical Society

Received: September 6, 2017 Accepted: November 30, 2017 Published: November 30, 2017 44281

DOI: 10.1021/acsami.7b13515 ACS Appl. Mater. Interfaces 2017, 9, 44281−44292

Research Article

ACS Applied Materials & Interfaces

Scheme 1. (a) Synthesis of Bifunctional Amphiphilic Block Copolymer PG-BPh; (b) PG-BPh Brush Coatings Fabricated via the “Adsorption-Cross-Linking” Approach Based on a Sequence of Versatile Photoinitiated C−H Insertion Cross-Linking (Scheme S1) Stepsa

Benzophenone (BPh) groups serve as hydrophobic domains in the first “Adsorption” step and further contribute to surface anchoring and/or intralayer cross-linking during UV irradiation in the second “cross-linking” step. The ω-N3 terminal groups in the bifunctional amphiphilic block copolymer serve as in situ modification sites. a

facilitated in obtaining a polyvalent anchoring on the substrates that lack aliphatic C−H groups (i.e., PTFE). The resulting PGBPh coatings showed excellent protein and cell resistance, which required and reversely indicated the high density of the polymer brushes, as described above. The antifouling performance was maintained for at least 1 year in the physiological buffer, which benefited from the stable anchoring and high thermal and oxidative stability of the polymer backbone. Although we have designed a set of mussel-inspired universal coatings in previous studies,20,21 the protein resistance on the coated hydrophobic nonpolar substrate (e.g., PS) surface never reached the same low level as that on TiO2 surface. The ongoing challenge of achieving such low fouling on nonpolar surfaces was only mastered in the present study, which allows the application of these antifouling coatings. Moreover, the PGBPh coating is “ready-to-use” for postmodification, profiting from the defined ω-terminal groups. Purposely designed N3 terminals were in situ functionalized with glyco-ligands, i.e., mannoses that on one hand, specifically adsorb concanavalin A (ConA), a kind of lectins, by multivalent protein−carbohydrate interactions and on the other hand, still prevent the nonspecific adsorption of other proteins.

challenge in constructing polymer brushes is obtaining a high grafting density, which results in lateral steric repulsion to stretch back-folded polymer chains into a brush conformation. All of the above-mentioned technologies often fail to generate brush conformation and hence do not reach a high protein resistance. Herein, we report a new polyglycerol (PG)-based bifunctional amphiphilic block copolymer with reactive anchors/ cross-linkers (Scheme 1) that rapidly generates a stable polymer coating on various nonpolar surfaces in a noninvasive manner. The hydrophobic property of benzophenone (BPh) was highlighted herein besides its well-known aliphatic C−H insertion chemistry. Benzophenone (BPh) has been widely used as a surface treatment reagent to tether polymer chains onto surfaces14,15 and photoinitiator to induce “grafting from” polymerization16,17 as well as cross-linker to generate surface gels.18,19 However, all of these applications only take place on the surfaces contain aliphatic C−H groups or require extra anchors, e.g., siloxane, to preimmobilize BPh on the substrates. Overall, BPh has never been recognized as a direct anchor on pristine surfaces. In this study, multiple BPhs were incorporated into the block copolymer as the cross-linkable hydrophobic block, which induced the hydrophobicity-based physisorption on various nonpolar substrates regardless of C−H groups and resulted in polymer brushes with high density even on poly(tetrafluoroethylene) (PTFE). In the second step, the BPh groups were covalently linked with substrates and/or neighboring polymer chains via photoinduced grafting and cross-linking (Scheme 1). The intralayer cross-linking combined with the weak hydrophobic interaction at the interface

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EXPERIMENTAL SECTION

Materials. All chemicals and solvents were reagents or HPLC grade, used as received, and purchased from Sigma (Steinheim, Germany) unless stated otherwise. The deionized water used was purified using a Millipore water purification system, with a minimum resistivity of 18.0 MΩ·cm. Allyl glycidyl ether (AGE) was purified by stirring over CaH2, distillation in vacuum before use, and storage over 44282


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For the online monitor of mannose surface immobilization, a precoated PS chip with PG-BPh was inserted into the flow chamber (QFM 401, Q-Sense, Sweden, internal volume of 40 μL) and incubated in MeOH with a flow rate of 0.05 mL/min. After baseline equilibration in the MeOH, a solution of Man−BCN (50 mg/mL in MeOH) was pumped into the flow chamber (0.05 mL/min). After 30 min of dynamic online adsorption, the flow chamber was rinsed with MeOH (0.1 mL/min). To ensure the complete reaction of the strainpromoted alkyne−azide cycloaddition (SPAAC), Man−BCN (50 mg/ mL in MeOH) was pumped into the flow chamber (0.05 mL/min) again for another 30 min and then followed with the buffer rinse. The whole measurement was performed at 25 °C. The conversion of this azide−alkyne cycloaddition at the interface was calculated from the equation

molecular sieves. Ethoxyethyl glycidyl ether (EEGE) was synthesized from 2,3-epoxypropan-1-ol (glycidol) and ethylvinyl ether according to a reference.22 It was purified by stirring over CaH2, distillated in vacuum before use, and stored over molecular sieves. The bicyclo[6.1.0]non-4-yn-9-ylmethyl(4-nitrophenyl)carbonate (BCN) was prepared according to a previously reported protocol.23 The syntheses of coating polymers and BCN-modified α-D-mannose are listed in the Supporting Information. Coating. Polystyrene (PS), poly(tetrafluoroethylene) (PTFE), polypropylene (PP), poly(vinyl chloride) (PVC), poly(ethylene terephthalate), polyurethane, and poly(dimethylsiloxane) (PDMS) slides (1 × 1 cm2) were cleaned ultrasonically in isopropanol and water. All of the quartz crystal microbalance (QCM) chips were cleaned according to the standard cleaning protocol from LOT (LOTQuantum Design GmbH, Darmstadt, Germany). To prepare PG brush coatings, the cleaned slides were immersed into a solution of 1 mg/mL PG-BPh in Milli-Q water at room temperature for 2 h. Thereafter, the slides were thoroughly rinsed with Milli-Q water and then dried by N2 stream. The coated slides were put under a light-emitting diode (LED) UV lamp (PLS-0365-010-11-C, 365 nm, 30 mW/cm2) and irradiated for 30 s. For the surface’s postmodification, cross-linked coating slides were dipped into a 10 mM Man−BCN methanol solution and shaken for 3 h at room temperature. The slides were thoroughly rinsed with methanol and Milli-Q water and then dried by N2 stream. It is important to note that the concentration of the polymer solutions for coating was always 1.0 mg/mL, which was far smaller than the critical micelle concentrations (CMCs) of PG-BPh polymer (>100 mg/mL). It has been shown that if the concentration of amphiphilic block copolymer was higher than the CMC, the block copolymer would form micelles in the selective solvent and the adsorbed surface layer itself may have a micellar structure instead of a smooth monolayer brush coating. The coating on the three-dimensional (3D) architectural surface was conducted in a similar operation as that of the coating on the planar surface. The fluorescein isothiocyanate (FITC)-modified PGBPh aqueous solution (5 mg/mL) was flowed into a pressure equalizer tube and a microfluidics chip and kept in the dark at room temperature for 2 h. The coating on platelet storage bags was obtained by immersing a piece of a platelet storage bag inside FITC-modified PGBPh solution and storing in the dark at room temperature for 2 h. Thereafter, we thoroughly washed them with methanol and Milli-Q water and then dried them by N2 stream. An LED UV lamp (365 nm; 42 mW/cm2) was used to initiate the cross-linking of benzophenone and further immobilization of the coating onto the 3D architecture surface. Quartz Crystal Microbalance (QCM) with Dissipation. Quartz crystal microbalance (QCM, Q-Sense E1, Sweden) with dissipation was used to test the adsorption on the sensor surfaces. QCM allows the monitoring of changes in resonance frequency (Δf) and dissipation (ΔD) of a piezoelectric quartz crystal as a function of time. f and D were recorded at the fundamental frequency (4.95 MHz) and its 3rd, 5th, 7th, 9th, 11th, and 13th overtones. Only the third overtone was shown in the sensor grams. The frequency response of QCM includes the contributions from both polymers and the water molecules that were bound to the polymer chains. Energy dissipation (D) changes represent the rigidity of the coatings, which is related to the hydration ratio and the conformations of the adsorbed polymers.24,25 For the adsorption of PG-BPh, the cleaned sensor chip was inserted into the flow chamber (QFM 401, Q-Sense, Sweden, internal volume of 40 μL) and incubated in Milli-Q water with a flow rate of 0.1 mL/ min. After baseline equilibration, a solution of PG-BPh (1 mg/mL in Milli-Q water) was pumped into the flow chamber (0.1 mL/min). After 1 h of dynamic online adsorption, the flow chamber was alternately rinsed with Milli-Q water, aqueous solution of deconex 1% (w/w, Borer Chemie AG, Switzerland), and Milli-Q water (0.1 mL/ min). The whole measurement was performed at 25 °C. The Sauerbrey equation was used to calculate the mass of the adsorbates (Δm = C × Δf, where Δm is the change in mass, C is the mass sensitivity constant of the quartz crystal (−17.7 ng/(cm2·Hz)), and Δf is the overtone-normalized frequency change).

conv =

mMan /MMan mcoating /MPG ‐ BPh

mcoating and mMan are calculated from the Sauerbrey equation. MPG‑BPh and MMan are the molecular weights of PG-BPh and Man−BCN, respectively. The protein adsorption was measured similarly. The coated sensors were inserted into the flow chamber and incubated in pH 7.4 N-(2hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES, 0.1 mol/ L, pH 7.4, and 150 mmol/L NaCl) buffer. After baseline equilibration, deconex (1% w/w in HEPES buffer) was pumped into the flow chamber for 10 min and followed by rinsing with HEPES buffer for 15 min. Then, the protein solution (1 mg/mL) was pumped into the flow chamber. After 30 min, the surface was rinsed with HEPES buffer again for another 15 min. The flow rate used for all experiments was 0.1 mL/min, and the temperature was 25 °C. The Voigt model for viscoelastic layers was used to calculate the mass of adsorbed proteins with the help of the software package Q-tools (version 3.0.15.553, QSense, Sweden) because the D/f ratio is not small enough to consider the surfaces as rigid surfaces. The density of the adsorbed protein layer was assumed to be 1200 kg/m3, the fluid density to be 1000 kg/m3, and the fluid viscosity to be 0.001 kg/ms. The proteins used to test the coating resistance were fibrinogen (Fib, 450 kDa, from bovine plasma), bovine serum albumin (BSA, 66 kDa), lysozyme (14.3 kDa, from chicken egg white), concanavalin A (ConA, 102 kDa, from Canavalia ensiformis), human blood plasma, and full fetal bovine serum (FBS) without dilution. Cell Resistance. Cell resistance experiments on PG-BPh-coated PP slides, PVC slides, and tissue culture polystyrene (TCPS) wells were done with adherent NIH-3T3 murine fibroblast cells (ACC no. 59, DSMZ, Braunschweig, Germany). Cells were collected from Petri dishes by incubation in trypsin (dilution 1:250) for 5 min at 37 °C. The trypsin was removed from cell suspension by centrifugation and the top layer was removed and the remaining cells were re-suspended in a fresh medium. The PP and PVC slides were incubated with 10 000 cells in 1 mL of cell medium (cell number was determined via a Neubauer chamber) for 3 days at 37 °C and 5% CO2. The wells of the 24-well cell culture plates were coated to test the coatings on TCPS. One milliliter of the cell medium with 10 000 cells was added into each coated/uncoated well. The incubated time was the same as that of the experiments with PP and PVC slides. The adhering cells were observed by a microscope directly (TELAVAL 31, Zeiss, Germany), without removing the medium or rinsing the slides with phosphatebuffered saline (PBS) buffer. Bacterial Specific Adhesion. For the adhesion experiments, a suspension of Escherichia coli (E. coli) (DH5α) in CASO broth with an optical density of 0.5, corresponding to about 6 × 108 bacteria/cm3 at the beginning of the exponential growth phase, was used. The control and modified PS and PTFE slides, 1.2 × 1.2 cm2, were washed with 70% ethanol, which was followed by five sterile PBS washes and then fitted in the bottom of a sterile 24-well plate. One milliliter of 1 × 106 CFU/mL of E. coli in a lysogeny broth medium was added to each well and incubated at 37 °C for 10 h without shaking. Each slide was then gently rinsed by immersion in sterile PBS four times. The bacteria adhered onto the substrate were stained by Syto 9 solution. After 10 44283


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Figure 1. (a) QCM frequency (f) shift as a function of time during the adsorption of PG-BPh on a PS sensor surface. The black curve shows buffer rinsing. The blue curve indicates physisorption of amphiphilic block copolymer, and the red curve demonstrates the surfactant washing. (b) Ellipsometric thickness of PG-BPh polymers on PS substrates with the evolution of the coating time. where s = σ(t)/σeq and T = t/τ are reduced variables, τ is a short characteristic time, and γ is a constant. In the terminal kinetic regime, polymer chains must penetrate a preexisting brush layer to reach the surface to tether onto the substrate. The change of grafting density in this regime is penetration-limited and predicted from the following relationship

min, the sample was gently immersed with fresh PBS buffer and observed after drying under confocal laser scanning microscopy (Leica DMI6000CSB SP8) with an FITC filter. Protein Adsorption from Mixed Proteins. The protein adsorption experiments on bare PS surface, PG-BPh coatings, and Man−PG-BPh coating surface were performed with BSA and ConA. The BSA was labeled with fluorescein isothiocyanate (BSA-FITC), and ConA was labeled with rhodamine B isothiocyanate (ConA-rhodamine B). The testing surfaces were incubated in the mixed solution of 1 mg/ mL BSA-FITC and 1 mg/mL ConA-rhodamine B in PBS buffer for 3 h at room temperature and then were gently rinsed with PBS buffer. The adsorption proteins on surfaces were directly observed by a fluorescent microscope (Axio Scope.A1, Zeiss, Germany) with FITC filter and DsRed filter. Coating Kinetics Study. The PS substrates used for the coating kinetics study were prepared by spin coating a homogeneous PS layer onto silicon wafers. Several drops of polystyrene dissolved in dimethylformamide (1% w/v) were dropped onto a clean silicon wafer and spin coated at 4000 rpm for 2 min. The thickness of the PS layer was 45 ± 0.2 nm determined by ellipsometry. The prepared PS substrates were then immersed into the PG-BPh polymer solution (1 mg/mL in H2O) for a set time. After rinsing with sufficient Milli-Q water and drying under high vacuum, the coating thickness was measured. According to the hypothesis of Ligoure and Leibler,26 the coating rate in the initial tethering regime (diffusion regime) was predicted to be controlled by the polymer chain diffusion from solution. The change of the coating density (below σ*) was proposed to follow the relationship below

σ(t ) ∼

⎛ Dt ⎞1/2 ⎜ ⎟ (ϕ0 /N ) ⎝ a2 ⎠

⎡ ⎛ t ⎞⎤ σ(t ) = σeq ⎢1 − exp⎜− ⎟⎥ ⎢⎣ ⎝ τex ⎠⎥⎦

τex is the exponential relaxation regime time (the penetration-limited regime).

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RESULTS AND DISCUSSION Synthesis of Amphiphilic Block Copolymer. The PGBPh bifunctional amphiphilic block copolymer was synthesized via the ring-opening anionic polymerization of ethoxyethyl glycidyl ether (EEGE) and allyl glycidyl ether (AGE), followed by acetal deprotection, thio-ene amination with cysteamine, and BPh linking by amide formation. The amount of the grafted BPhs was verified to guarantee an optimal coating density and brush conformation of the adsorbed polymers from aqueous solution (1 mg/mL). With the increase of BPh content in the polymer chain, the thickness of the resulting coatings on PS surface gradually increased and reached a plateau value at approx. 3.2% (in mole ratio) BPh content, corresponding to ∼4 BPh units per polymer chain. At this BPh content, the water contact angle (WCA) plot exhibited an inflection point. The surface hydrophobicity decreased before reaching this point and increased hereafter (Figure S1). This indicates that a change of the internal layer structure occurs at this point (water contact angle of 63 ± 1°) and suggests that the layer termination is dominated by PG-based chains. We conclude that a certain amount of BPh was required to generate enough hydrophobicity at the interface, but too many BPh units would increase the steric effect and result in a less dense coating. Therefore, in the following, the block copolymer with 4 BPh units was utilized unless otherwise specified. In a block-selective solvent (H2O), which is good for the PG block but poor for the BPh block, pristine substrate surfaces were exposed and an adsorbate layer was formed. Quartz crystal microbalance (QCM) with dissipation was employed to analyze

(1)

where σ(t) is the polymer graft density, D is the diffusion coefficient of the polymer, and ϕ0 is the volume fraction of monomer in solution. The σ* denotes the grafting density at which the tethered chains just start to overlap (σ = σ* ≈ N−1). Above the overlap concentration, a further adsorption requires some stretching of the chains. There is a potential barrier to oppose the penetration of the chains. The barrier becomes an essential obstacle, and the diffusion of free chains does not control anymore the adsorption kinetics. The crossover surface coverage σ is defined as27 s1/3 eγNσeq

2/3 2/3

s

≅

2 Nσeq 2/3T 3

(3)

(2) 44284


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Figure 2. (a) Deconvoluted XPS S 2s signal curves of PG-BPh coatings on PS substrate and the corresponding curves of pristine PS substrate; (b) the average surface roughness (Rq and Ra) for bare and coated PS surfaces, which was calculated from five atomic force microscopy (AFM) images (2 × 2 μm2); (c) the image of the microfluidic chip (PDMS); and (d) the corresponding fluorescence image after PG-BPh coating. The PG-BPh polymer used here was covalently modified with FITC isothiocyanate (2% to all OH groups in the backbone) prior to coating.

onto the PS substrate and/or completely cross-link the polymer chains. The BPhs were only located in the hydrophobic block of the copolymers; thus, the intralayer cross-linking did not affect the brush conformation of the hydrophilic PG blocks that were oriented toward the substrate surface. The first step of polymer diffusion onto the solid substrates allowed a very fast layer preparation/formation. As indicated by ellipsometry (Figure 1b), the dry thickness reached a plateau value with approx. 3.5 nm in 3 min. This value did not increase even after immersion time up to 10 h, which matched the change of hydrated thickness obtained by Voigt fitting from QCM curves (Figure S3). We concluded that the respective surface coverage corresponds to the equilibrium surface grafting density. The grafting density of polymer chains was calculated according to the following equation29

the adsorption of the polymers on PS sensors. The results show that the hydrated mass of the adsorbed PG-BPh on the sensor surfaces was about 950 ng/cm2 (Figure 1). However, the polymers only weakly adsorbed onto the PS substrate and were easily rinsed away by surfactant solutions. Nearly 65% of the adsorbed polymers detached after rinsing with 1% w/w deconex aqueous solution. This result was expected and agreed with the properties of adsorbates formed by traditional amphiphilic polymers. However, when the freshly deposited coatings were exposed to UV irradiation (365 nm, 42 mW/ cm2), the obtained coatings maintained stability upon rinsing with surfactant solutions (Figure 1a). Under UV irradiation, the BPh groups underwent an n−π* transition into a triplet state and abstracted a hydrogen atom from neighboring aliphatic C− H groups, i.e., from either the substrate or the neighboring polymer chains,28 which resulted in a covalent C−C bonding with the substrate or the neighboring polymer chain (Schemes 1 and S2). The optimized UV irradiation time was identified by a decrease in WCA and increase in coating stability under subsequent rinsing with deconex (Figure S2). Only 30 s of irradiation was sufficient to covalently immobilize the polymers

σ = (hρNa)/M n

where h is the dry thickness of the coating, ρ is the bulk density of the coating polymer (assumed to be 1.1 g/cm3 for PG-BPh), Na is the Avogadro number, and Mn is the number average 44285


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Figure 3. (a) Grafting density of PG-BPh polymers as a function of t0.5 in the diffusion-controlled region. (b) ln(t) dependence of the grafting density of PG-BPh in the crossover regime. (c) Natural logarithm of the normalized grafting density of PG-BPh as a function of time in the terminal regime (d) ΔD−Δf plots of the adsorption of PG-BPh on PS sensor surfaces (60 min online coating). The starting point of the dynamic coating process was defined as ΔD = Δf = t = 0.

molecular weight of the polymer. The resulting equilibrium coating density was about 0.2 chains/nm2, which was in the scale of a dense monolayer brush coating and significantly higher than previously reported “grafting to” brush coatings.30,31 As mentioned above, the generated radicals from BPhs can randomly insert any neighboring aliphatic C−H group and form covalent C−C bonds, regardless of the coating polymer and the substrate. Therefore, the PG-BPh coatings displayed substrate-independent properties. The static water contact angles of all of the tested surfaces significantly decreased after the coating, as expected (Figure S4). Following the UV treatment, the PG-BPh polymers were even effectively tethered onto chemically inert PDMS and PTFE, which are otherwise hard to coat. The stability of the PG-BPh coatings was investigated on selected substrates, namely PS, PP, and PTFE. The contact angles of the PG-BPh-coated surfaces did not obviously increase after incubation in a sodium dodecyl sulfate solution (2%, w/w) for 30 min under sonication (Figure S5). The chemical composition of PG-BPh coatings on PS substrates was confirmed by X-ray photoelectron spectroscopy (XPS) analysis (Table S1). The deconvoluted S 2s spectra for PG-BPh coating and the corresponding pristine PS substrate is shown in Figure 2a. The significant S 2s peaks, ascribed to the

sulfur of sulfide bond, were only detected in the case of substrates covered with PG-BPh polymer coatings but not in the case of pristine substrates (Table S1); this finding confirms the successful polymer coating. The corresponding N 1s spectra was shown in Figure S6. The nitrogen peaks at 399.0 ± 0.1 and 401 ± 0.1 eV were attributed to the nitrogen (N−H, amide) and cationic ammonium nitrogen in the polymer chain, respectively. It was assumed that the only peak at ∼399 ± 0.1 eV in the pristine substrate spectrum related to the N−H bond of some additive in PS. The surface morphology of the coatings and the respective bare substrates was investigated by atomic force microscopy (AFM) (Figure S7). On the coated substrates, island-like structures could be clearly observed and this morphology was construed typical of monolayer polymer brush coating.32 The monolayer coatings were ultrasmooth and ultrathin. Both the average roughness (Ra) and root-mean-squared roughness (Rq) of the coatings were slightly smaller than those of the bare PS substrate (Figure 2b). We assume that the coating levels the grooves of the substrates. The coating thickness under ambient condition was 3.6 ± 1.0 nm, as measured by AFM, and correlated well with the ellipsometry measurements (Figure 1b). 44286


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Figure 4. (a) Protein adsorption on PG-BPh-coated PS surfaces. (b) Protein adsorption from FBS to the coated PS surfaces before and after incubation in PBS buffer for 1 month, 2 months, 6 months, and 1 year. (c) NIH-3T3 cell adhesion on the PG-BPh-coated PS and the pristine surfaces after 3 days of cell culture. Scale bar: 50 μm.

characteristic time τ1 is obtained from the inflection point of the fitting curve and indicates the end of the first kinetic regime. The corresponding areal density of polymer chains on the substrate surface is σ1. The average distance between tethering sides on the substrate was defined as d, which can be calculated from the experimental values of σ following the equation: d = (σπ/4)−1/2.33 The value of d at the end of the diffusion regime was 7.7 nm, which is far larger than that of the gyration radius (Rg) of PG-BPh polymers (that is 1.7−1.8 nm for polyglycerol with molecular weight 10 kDa35). According to the commonly used criterion,33 it may be expected that for d > 2Rg, the tethered polymer chains have sufficient lateral space on the substrate surface and should be in an expanded coil or mushroom conformation. Therefore, a layer of nonoverlapping and relaxed polymer chains covered the substrate in the first coating regime. When the grafting density further increased, the tethering rate progressively slowed down and was proportional to ln(t) due to the increased energy barrier (Figure 3b). This coating regime was controlled by the diffusion of free polymers through the already tethered chains to reach the substrate surface. This

Besides the coating on two-dimensional (2D) planar surfaces, the polymers can also be efficiently used for the complex 3D systems, including PDMS microfluidic chips (Figure 2c,d), polyethylene microtubes, as well as PVC blood platelet storage bags (Figure S8), by simple dip-coating. Besides, PG-BPh coatings were also successful on polar inorganic surfaces (including gold, titania, and silica) because of the cross-linkable BPh anchors (Table S2, Figure S9). Kinetics of Brush Formation. The adsorption of polymer chains from dilute solution to the impenetrable solid surface to form a monolayer of polymer brushes has been extensively studied.27,33,34 According to the hypothesis of Ligoure and Leibler,26 the initial tethering regime (diffusion regime) for an end-functionalized polymer attached to a substrate surface should be diffusion-controlled and exhibit a t0.5 dependence on the tethering time (eq 1). The diffusion of polymer chains from solution to the substrate surface is comparably fast. The tethering rate of PG-BPh onto a PS substrate surface in the diffusion regime corresponded well to the prediction, as shown in Figure 3a. During the first 1.3 min, the surface grafting density (σ) exhibited a linear relationship with t0.5. The 44287


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corresponded to t = 1.3 min (τ′1) and exactly matched the end time for the diffusion-controlling regime. Following the second phase, a third phase with a smaller slope (|∂D/∂f 3| = 0.02) appeared where ΔD only slightly increased with an increase of |Δf |. The adsorbed polymers in this phase were likely to compact the coating in such a way that the trapped water was replaced by additional polymer chains,25 which resulted in highly dense coatings. The break point (τ′2) between these two phases was at 22 min, which coincided with the onset time of the penetration-limited regime as well. Antifouling Performance. After successful monolayer characterization, the protein resistance of the PG-BPh coating was evaluated by single proteins including fibrinogen (Fib), bovine serum albumin (BSA), and lysozyme (Lys), as well as by the complex protein environment of undiluted fetal bovine serum (FBS) and human blood plasma. As indicated by QCM studies, the coatings exhibited an extremely high protein resistance (Figure 4a, Table S3). The coated PS surface successfully repelled >97% of the adsorbed single proteins relative to the bare PS surfaces. More notably, Fib, a large protein present in relatively large quantities in the blood and strongly adsorbing to hydrophobic surfaces, was repelled till

High-antifouling Polymer Brush Coatings on Nonpolar Surfaces via

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